Self-assembling membranes

Self-assembling polypeptide membranes with adjustable pore properties

We investigated potentials of fusing protein oligomerization domain with coiled-coil-forming domain for the formation of nanostructures. This combination should form a two- or three-dimensional lattice if the coiled-coil-forming domain forms an antiparallel homodimer. Particular advantage of this approach is that we can incrementally extend or modify the coiled coil-forming domain, which results in modification of the size of the lattice unit as well as in the size of pores enclosed by coiled-coil segments.

We designed and prepared fusion protein between p53 tetramerization domain and a homodimeric antiparallel coiled-coil domain. This type of nanomaterial contains nanopores of defined size, which could be used to separate molecules or molecular assemblies according to their size. We assembled the material by a refolding procedure and tested it in a real world application as ultrafiltration membrane. The membranes efficiently remove large molecules and viruses from the sample.

Figure 1: The summary of the idea of self-assembled polypeptide membrane and its performance in removing viruses from solution. A) The polypeptide material composed of tetramerization domain linked to coiled-coil-forming domain . B) The removal of viruses from the solution with the self-assembled polypeptide membrane.

The idea and approach

The self-assembly of nanoBricks-like modular proteins composed of different association-prone domains should in theory lead to formation of polypeptide assemblies. When at least one of the association-prone domains forms a trimer, tetramer or higher oligomeric species, formed polypeptide material can assemble in two or three dimensions. The possible applicability of such materials is enormous; it could be used for biosensors, chemical catalysis, drug delivery, crystallization etc.

Our idea was to design polypeptide materials of oligomerization domain and coiled-coil-forming domain, where the oligomerization state is preferably three, four or more. Antiparallel coiled-coil segments of such fusion polypeptides would tether the oligomerized hubs of protein domains creating a polypeptide lattice (Figure 2). This type of assembly would be especially useful since materials with different physicochemical properties could be formed with minor variations in building blocks. While there are multiple possible uses of such materials, we focused on its filtration properties. Membrane-based filtration systems are widely used in preparation of drinking water, for removal of water pollutants, infectious microorganisms and for concentration of selected components, and are gaining importance in food industry. Ultrafiltration membranes are typically made of organic polymers, however, we demonstrate the feasibility of manufacturing a membrane from self-assembling polypeptide nanobricks. The advantage of our technique is that the molecules self-assemble in a desired fashion and the size and properties of the pores depend on the properties of its building blocks.

Figure 2: Assembly of a polypeptide consisting of a tetramerization domain and antiparallel homodimeric coiled-coil-forming domain results in a lattice with pores (circles) of defined size and properties depending on the nature of the coiled-coil segment.

In order to test the formation and applicability of such designed membranes we prepared fusion proteins where oligomerization-prone domain is a tetramerization domain of p53 and a coiled-coil-forming segment is either a designed antiparallel homodimeric coiled-coil APH, APH1 (Gurnon, 2003) or BCR, a coiled-coil forming domain from the natural protein (Taylor, 2005). All of selected coiled-coil-forming domains associate into antiparallel coiled-coil dimers but they differ in length (45, 31 or 36 amino acid residues) and stability (Figure 3). We demonstrate that membranes formed by self-assembly of nanoBricks successfully retained large molecules and viruses when filtration was performed

Figure 3: Scheme of the constructs (A) and 3D model of APH-p53 fusion construct (B).

• Results